Adherence of pathogens to their host cells is the obligatory first step of infection and is frequently mediated by specific molecular interactions [1][2]. Virulent Campylobacter species, Vibrio cholerae, enteropathogenic E.coli (EPEC), enterohemorrhagic E.coli (EHEC) and pathogenic strains of Norwalk virus, the leading bacterial and viral causes of human infectious diarrhea [3], adhere to gut epithelial surfaces through binding to 1(1,2) fucosylated cellular receptors[4][5]. 1(1,2) fucosylated glycans, which are abundant in human breast milk[6][7], have been shown both in vitro and in vivo effectively to prevent binding and infection by these pathogens[4][5]. These molecules therefore represent a new class of agent with potential to prevent infectious diarrhea, a condition that is the cause annually of over 2 million deaths worldwide [8]. However the production of 1(1,2) fucosylated glycans as anti-infective agents in sufficient quantities to impact global diarrhea incidence remains a significant challenge. Chemical syntheses are possible, but are limited by stereo-specificity issues, product impurities, and high overall cost[9][10][11]. In vitro enzymatic syntheses are also possible but are limited by a requirement for expensive nucleotide-sugar precursors. Glycosyn Inc.<s broad goal is to develop ways to manufacture 1(1,2) fucosylated glycans cheaply and in bulk through microbial fermentation, and three classes of potential anti-infective products are envisaged: 1) purified 1(1,2) fucosylated oligosaccharides, 2) yeast strains expressing 1(1,2) fucosylated glycans on their cell surface, and 3) purified 1(1,2) fucosylated glycoproteins. [[[The goal of the studies outlined in this application are to produce in the dairy yeast Kluyveromyces lactis an example of the first of these product classes, namely a purified 1(1,2) fucosylated oligosaccharide, 22-fucosyllactose (22-FL), in sufficient amounts to test this molecule<s efficacy as a single agent in in vitro and in vivo infection models. Glycan synthetic pathways in Kluyveromyces lactis will be engineered through a combination of endogenous gene manipulation and the introduction of heterologous genes encoding desired activities. Specifically, K.lactis will be engineered to synthesize the key precursor sugar, GDP-fucose, and subsequently to make 22-fucosyllactose in the cell cytoplasm. A subsequent goal is to increase the yield of 2<-fucolsyllactose recovered from K.lactis to approach levels that will be required for commercialization. To achieve this the production of 2<fucosyllactose will be increased by manipulating cellular levels of synthetic enzymes and precursor pools, balancing 2;-FL production with overall cell viability and growth performance under bioreactor conditions.]]]